Project Summary Proper gene network connectivity is essential for regulating gene expression, yet the rewiring of gene regulatory networks is crucial for the evolution of phenotypic innovations. Although progress has been made in mapping the components and deciphering the function of these networks, the mechanisms by which such intricate circuits originate and evolve remain poorly understood. It has been hypothesized that domestication of transposable elements (TEs) could allow identical regulatory motif(s) to be recruited to numerous genomic locations and rapidly draw multiple genes into the same regulatory network. However, the rapid decay of domesticated TEs make it extremely difficult to recognize ancient elements and assess their contribution to non-coding regulatory sequences. Recently, the Bachtrog lab provided the first direct, functional evidence for the rewiring of a gene regulatory network via TE domestication by showing that dozens of novel binding sites for the dosage compensation complex along the neo-X chromosome in Drosophila miranda originated from the domestication of a helitron TE. Here, I propose to leverage the naturally-occurring variation in Drosophila sex chromosome karyotypes to characterize independent replicates of a major gene regulatory network rewiring event: the evolution of dosage compensation on a newly formed neo-X chromosome. I will use phylogenetic dating and gene divergence to date the neo-sex chromosome fusion event for multiple Drosophila species with neo-sex chromosomes. Transcriptomic analyses will determine the extent to which dosage compensation has evolved on the neo-X chromosomes. Informative neo-sex chromosomes of intermediate age (~1-10 MYA) that have evolved at least partial dosage compensation will be used in downstream experiments and analyses. ChIRP-seq of dosage compensation-specific roX RNAs will be used to identify novel dosage compensation chromatin entry sites (CES) on the independently formed neo- X chromosomes. Comparison of CES sequences with homologous regions in closely related taxa that lack neo-sex chromosomes will be used to characterize the mutational paths of novel dosage compensation binding site evolution. The project addresses key questions in gene network regulation and sex chromosome evolution, such as how repeatable is molecular evolution, how essential is TE domestication in rewiring regulatory networks, and how does the evolution of dosage compensation correlate with Y chromosome degeneration? The biological insight obtained from this analysis has broad implications for the understanding of gene expression regulation, molecular evolution, and sex chromosome evolution.